The fascinating process by which proteins fold to complex 3-dimensional structures plays critical roles in their myriad functions in cells and organisms, and in misfolding that can lead to disease. Protein misfolding and aggregation (amyloid formation) are implicated in several diseases such as Parkinson's and Alzheimer's diseases, prion diseases, diabetes, and heart disease. Furthermore, recent work supports the intriguing idea that protein amyloids can also have functional roles in biology. Therefore, understanding the mechanisms by which proteins fold is critical to furthering our understanding of basic biology and disease, understanding that could later assist with design of therapeutic strategies in this regard. While much has been learned about protein folding and aggregation, these are extremely complex processes, leaving a host of issues yet to be resolved. One aspect of recent note along these lines is the increasing realization that a spectrum of protein disorder (flexibility) is prevalent in genomes, and that this disorder is often intimately linked to functio and malfunction. For example, monomeric forms of several amyloid-forming proteins have extensive stretches of disordered sequence. Here, we propose to develop and apply novel single-molecule fluorescence methods to take next key steps in furthering our understanding of several of the above aspects. Our work will focus on the Parkinson's disease-linked protein ?-synuclein, which has multiple putative biological functions. This protein has substantial disorder as a monomer and can fold upon binding to partners, an interesting feature that it shares with many disordered proteins. Although ?-synuclein has been investigated for many years, limited insight has been gained about its complex and dynamic biophysics. By avoiding the averaging inherent in most ensemble experiments, we will probe the complex folding landscape of this system and its natural variants during interactions and aggregation at a resolution not feasible by standard methods. Our innovative work will uniquely combine facets of cutting-edge biophysics, optics, biochemistry, and chemical biology. Overall, the work will result in a new level of understanding of the ?-synuclein system, with implications for better understanding of the biology of neurodegenerative diseases. Furthermore, we will develop and implement combination, state-of-the-art single-molecule tools which will be broadly applicable in studies of a host of other biologically important molecules.